Immobilized metal ion affinity chromatography (IMAC) is a technique used for the purification of proteins. The technique is based on the natural ability of some proteins to bind to transition metals. Porath, J., Carlsson, J., Olsson, I., and Belfrage, G. (1975) Nature 258 598-599. The affinity of proteins for transition metals derives from the properties of amino acids within the protein's primary structure which interact with and bind to metal ions. However, the occurrence of such metal/protein binding is random, due to the fact that not all proteins possess such metal binding amino acid sequences. Moreover, the strength of the bond to the metal ion varies unpredictably with the particular protein. Furthermore, if two or more protein molecules in a given mixture possess such metal binding sequences, the usefulness of the technique as a purification method is diminished since both will bind to the immobilized metal ion.
These shortcomings of the IMAC technique were solved by the advent of the CP-IMAC technique which provided a predictable and specific method for the purification of proteins by exploiting this natural protein-metal binding phenomenon. The term "CP-IMAC" reflects the use of "chelating peptides" to specifically bind immobilized metal ions and purify proteins which contain such chelating peptides via IMAC principles. Chelating peptides are short amino acid sequences which are specifically designed to interact with and bind to metal ions. Smith, M. C., Furman, T.C., Ingolia, T.D., Pidgeon, C. (1988) J. Biol. Chem. 263, 7211-7215. A protein possessing a chelating peptide (herein termed a CP-protein) will bind to an immobilized metal ion with high specificity in a relatively stable manner. This allows one to remove all nonspecifically or weakly bound proteins from the column and later isolate the purified CP-protein. If desired, the CP-protein may then be enzymatically or chemically treated to remove the chelating peptide leaving only the purified protein of interest. However, the utility of CP-IMAC is restricted by the transient nature of the CP-protein/metal complex. The utility of chelating peptides in conjunction with proteins could be expanded to many other applications if one were able to more permanently bind the protein to the support matrix.
The instant invention describes a reversible method of forming a kinetically inert complex between a CP-protein and a metal ion by binding the CP-protein to the metal ion through CP-IMAC methodology and subsequently varying the oxidation state of the metal ion so as to convert the labile complex used in the CP-IMAC procedure to a kinetically inert complex.